Showing papers on "Sperm plasma membrane published in 1997"

Reactive oxygen species and sperm physiology

Abstract: Although high concentrations of reactive oxygen species (ROS) cause sperm pathology (ATP depletion leading to insufficient axonemal phosphorylation, lipid peroxidation and loss of motility and viability), recent evidence demonstrates that low and controlled concentrations of these ROS play an important role in sperm physiology. Reactive oxygen species, such as the superoxide anion, hydrogen peroxide and nitric oxide, induce sperm hyperactivation, capacitation or the acrosome reaction in vitro. The ROS involved in these processes may vary depending on experimental conditions, but all the evidence converges to describe these events as ‘oxidative’ or ‘redox regulated’. Human sperm capacitation and acrosome reaction are associated with extracellular production of a superoxide anion that is thought to originate from a membrane ‘oxidase’. The enzymes responsible for tyrosine phosphorylation‐dephosphorylation of sperm proteins are possible targets for ROS since mild oxidative conditions cause increases in protein tyrosine phosphorylation and acrosome reaction. The lipid peroxidation resulting from low concentrations of ROS promotes binding to the zona pellucida and may trigger the release of unesterified fatty acids from the sperm plasma membrane. The fine balance between ROS production and scavenging, as well as the right timing and site for ROS production are of paramount importance for acquisition of fertilizing ability.

Why did the sperm cross the cumulus? To get to the oocyte. Functions of the sperm surface proteins PH-20 and fertilin in arriving at, and fusing with, the egg.

Abstract: The sperm surface has an active role in the events of fertilization. The definition of the sperm surface in both its composition and domain organization begins during spermatogenesis and continues until the moment of sperm-egg fusion. Alterations of the surface proceed as a result of internal programming and environmental cues from both the male and female reproductive tracts, including interactions with the egg itself. We have investigated the sperm surface to understand its domain organization and the ongoing changes in this organization as well as the role of specific surface proteins in fertilization. Much of our research has concentrated on two surface proteins: PH-20 and fertilin. PH-20 is a single-chain protein, anchored in the membrane via a glycosyl phosphatidylinositol (GPI) anchor. The N-terminal domain of the molecule has a hyaluronidase activity. The hyaluronidase activity of PH-20 on the sperm plasma membrane enables sperm to penetrate the layer of cumulus cells surrounding the oocyte. PH-20 has a second function, unrelated to its hyaluronidase activity, in the binding of acrosome-reacted sperm to the zona pellucida (secondary sperm-zona binding). The fertilin molecule is an alpha,beta heterodimer whose two subunits are closely related transmembrane proteins. Fertilin beta has a disintegrin domain that has high sequence homology with the snake disintegrins, a known class of soluble integrin ligands. The binding site of the beta disintegrin domain functions to bind sperm to the egg plasma membrane via a mechanism that leads to sperm-egg fusion. The precursor of fertilin alpha, made in the testis, has an active metalloprotease site that could function in spermatogenesis. This metalloprotease domain is removed by proteolytic processing in the testis. Mature fertilin alpha on sperm also has a hydrophobic, putative "fusion peptide" that may promote the process of lipid bilayer fusion between sperm and egg plasma membranes. Fertilin alpha and beta are the first identified members of a new gene family of transmembrane proteins, the ADAM family, so called because they contain A Disintegrin And Metalloprotease domain. Many distinct ADAMs have now been found in diverse tissues and species (Drosophila to human) and are proposed to have a variety of functions in development and the adult. In addition to fertilin, other ADAMs are also present on the sperm plasma membrane and may participate with fertilin in sperm-egg fusion.

Sperm plasma membrane remodeling during spermiogenetic maturation in men: relationship among plasma membrane beta 1,4-galactosyltransferase, cytoplasmic creatine phosphokinase, and creatine phosphokinase isoform ratios.

Abstract: Sperm creatine phosphokinase (CK) concentrations and the synthesis of the CK-M isoform reflect normal spermiogenesis and predict maturity and fertilizing potential of ejaculated human spermatozoa. Immature spermatozoa, characterized by cytoplasmic retention and low CK-M to CK-B isoform ratios, are deficient in zona binding and fail to cause pregnancies. Because these sperm lack zona-binding ability, we examined in this study whether beta 1,4-galactosyltransferase (GalTase), a key element of sperm-zona interactions in mice, is diminished in immature human sperm. Unexpectedly, GalTase was overexpressed in immature sperm relative to mature sperm: the levels of cytoplasmic CK and plasma membrane GalTase were positively correlated (r = 0.78, p < 0.001, n = 88). Sperm populations with various levels of cellular maturity, prepared by Percoll gradients, had different CK and GalTase concentrations, but within each subpopulation the relationship between CK and GalTase was maintained (p < 0.01-0.001). GalTase activities in intact and vortex-disrupted sperm fractions were similar, showing that GalTase is present on the surface membrane of human sperm--similar to the situation in all other species assayed. The changes previously reported by our laboratory in zona-binding ability and lipid peroxidation rates (which occur simultaneously with cytoplasmic extrusion), decline in CK activity, and increased expression of the CK-M isoform are suggestive of a remodeling of the sperm surface concomitant with cytoplasmic maturation. The changes reported here in GalTase expression on the surface of maturing spermatozoa prove this hypothesis.

Fundamental Cryobiology of Mammalian Spermatozoa

Abstract: Publisher Summary This chapter discusses the fundamental cryobiology of mammalian spermatozoa. The low motility of cryopreserved mammalian spermatozoa and the often lower conception rates may be due to the fact that procedures for cryopreservation of many mammalian cell types, including sperm, have evolved empirically. The primary assay of sperm function is the use of insemination and measurement of pregnancy initiation. The approaches to measuring sperm plasma membrane integrity include supervital staining and hyposmotic swelling. The other general approach to evaluating plasma membrane integrity is to assay the maintenance of membrane semipermeability by testing the cell's ability to change its volume when exposed to anisomotic conditions. Survival of cells subjected to cryopreservation depends not only on the presence of a permeating cryoprotective agent (CPA) but also on the concentration of the CPA. Viability of mammalian sperm is very sensitive to osmotic stress and the associated cell volume excursion. The optimization of CPA addition and removal procedures is also elaborated.

Regulation of membrane stability and the acrosome reaction in mammalian sperm.

Abstract: Membrane destabilization is an essential step in the process of membrane fusion. In many cell types, exocytotic fusion may occur sporadically at microscopically localized sites on the surface of the cell, making it difficult to study the chemical and physical features of the membrane (or membranes) that promote fusion. In the sperm cell, exocytosis occurs synchronously at a distinct region on the sperm head. This localization of function makes the sperm cell a useful model to investigate the structural features of the bilayers that control membrane fusion. During sperm maturation, the anterior head membranes undergo a well-defined series of chemical, physical, and functional changes that are necessary to produce a fertile gamete. These changes include the addition of highly unsaturated phosphatidylcholine, a decrease in general membrane stability, and an increase in the ability to respond to physiological and pharmacological inducers of exocytosis. Concomitant addition of cholesterol and an actively maintained asymmetric transmembrane phospholipid distribution modulate these effects to stabilize the membrane of the mature sperm for storage. The environment of the female tract provides conditions that promote efflux of cholesterol from the sperm plasma membrane as well as the loss of membrane asymmetry. The cholesterol-poor, lipid-symmetric plasma membrane has a destabilized inner leaflet that facilitates membrane fusion upon binding of the sperm to the appropriate egg coat receptors. We summarize these features in a mechanistic model in which the sperm membrane contains destabilizing components to confer fusogenic potential as well as stabilizing components organized to maximize membrane integrity. This combination prevents premature fusion in the male tract. After deposition in the female tract and removal of the stabilizing components, followed by reorganization of the fusogenic components, the membrane becomes poised to fuse upon receipt of the final biological stimulus.

Changes in calcium transport in mammalian sperm mitochondria and plasma membranes caused by 780 nm irradiation.

Abstract: Background and Objective Regulation of intracellular Ca2+ concentrations are very important in control of sperm motility and acrosome reaction. It was shown previously that low-power lasers in the visible and near-infrared range alter Ca2+ uptake by sperm cells. In the present work the effect of a 780 nm diode laser on Ca2+ uptake by sperm mitochondria and isolated plasma membrane vesicles is investigated. Study Design/Materials and Methods Digitonin-treated spermatozoa and plasma membrane vesicles were irradiated with a 780-nm diode laser at various powers and energy doses, and Ca2+ uptake was measured by the filtration method. Results It was found that 780-nm irradiation inhibits Ca2+ uptake by the mitochondria but stimulates Ca2+ binding by sperm plasma membrane vesicles. The effect of light on Ca2+ uptake by plasma membrane vesicles in the absence of ATP was much larger than that measured in the presence of ATP. Addition of Ca2+ ionophore decreased the Ca2+ uptake by the irradiated membranes in the presence of ATP but enhanced it significantly in the absence of ATP. Conclusion 780 nm light inhibits Ca2+ uptake by sperm mitochondria and enhances Ca2+ binding to sperm plasma membranes. Lasers Surg. Med. 21:493–499, 1997. © 1997 Wiley-Liss, Inc.

Sperm-associated oocyte-activating factor is released from the spermatozoon within 30 minutes after injection as a result of the sperm-oocyte interaction.

Abstract: We have previously shown that sperm plasma membrane damage makes the sperm plasma membrane permeable and the sperm nucleus accessible for low molecular weight molecules such as eosin and dithiothreitol. In the present study, we investigated whether this damage is associated with a passive release of the sperm-associated oocyte activating factor (SAOAF) from the spermatozoon and, if so, its time sequence. In a first study, human oocytes remaining unfertilized after conventional in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) and freshly ovulated mouse oocytes were injected with a whole spermatozoon or a sperm head respectively. They were randomly allocated to one of three groups: oocytes in group 1 were injected with a spermatozoon immobilized or sperm head detached immediately prior to the injection; oocytes in group 2 were injected with a spermatozoon immobilized or sperm head detached 2-4 h before injection; oocytes in group 3 were injected with a spermatozoon or sperm head that had been subjected to heat treatment. The activation rate of oocytes injected with a spermatozoon or sperm head was the same for groups 1 and 2, and significantly higher than in group 3 (P < 0.001). In a second series of experiments, human oocytes remaining unfertilized after IVF or ICSI were injected with a sperm head that was subsequently removed from the ooplasm 20-30 min after injection. The activation rates were compared to that of oocytes injected with heat-treated spermatozoa which subsequently were removed from the ooplasm. We found that the removal of the spermatozoon 30 min after injection did not prevent oocyte activation. Our data indicate that the initial damage to the sperm plasma membrane induced at immobilization, although essential for the onset of sperm nuclear swelling after ICSI, does not by itself lead to the release of SAOAF from the spermatozoon. We postulate, however, that SAOAF is released during the sperm nuclear swelling phase, which is induced by the so-called sperm nucleus decondensing factor (SNDF) of the oocyte.

Sperm plasma membrane characteristics and boar semen fertility.

Abstract: Much effort is being made to establish relationships between the molecular events that take place in spermatozoa under fertilizing conditions and actual sperm function during fertilization. During capacitation, the process that 'primes' spermatozoa for interaction with the egg, components of the sperm's environment, notably bicarbonate, provoke various specific changes in the architecture and functioning of the sperm plasma membrane in a large number of cells. The individual changes have been found to proceed on different time scales, and may therefore represent sequential stages in the capacitation process. However, each change takes place at different rates in individual cells, revealing considerable functional heterogeneity within the sperm population. Recent work on membrane changes provoked by cooling has indicated similarities with capacitational changes. The effect of cooling may therefore be to induce premature capacitation (and destabilization). Such an effect would greatly compromise sperm fertilizing potential. A pig sperm-egg interaction model was used to examine quantitative details of zona binding and zona penetrating abilities within capacitated sperm populations, and sperm behaviour was found not to accord with generally held beliefs. In particular, individual spermatozoa that have bound to the zona pellucida show great variation in the delay before penetrating: no evidence has been found for a specially competent subgroup. Even in sperm samples incubated to undergo maximal capacitational membrane changes, cells with actual penetrating potential represent less than 15% of the total number that attach initially to the zona pellucida. Thus detection of capacitational membrane changes appears greatly to overestimate zona penetrating capability. Future studies linking sperm membrane characteristics with semen fertility in the field will need to consider differences between in vitro and in vivo conditions. The need for survival in the female tract may require much slower sperm responses than are considered optimal for in vitro fertilization.

Reversible contraceptive effect of PH-20 immunization in male guinea pigs.

Abstract: Sperm proteins are currently being studied as antigens on which to base a contraceptive vaccine. Sperm plasma membrane proteins offer the theoretical possibility of immunizing either males or females and achieving a contraceptive effect. In this study, we investigated the sperm plasma membrane protein PH-20 as an antigen for inducing infertility in males. We found that infertility can reproducibly be induced in male guinea pigs immunized with purified PH-20: 100% (29 of 29) of PH-20-immunized males became infertile, whereas all 22 controls were fertile. The males were extremely responsive to PH-20 immunization: infertility could be induced with a single injection of only 5 microg PH-20. Among males that received their initial injection when they were approximately 300 g (body weight), 14 of 15 had regained fertility at about 1 yr after initial injection. Surprisingly, in another group of males that received their first injection when they were approximately 650 g (body weight), only 1 of 5 had regained fertility about 1 yr after initial injection. Anti-PH-20 titers in antisera (2 mo after initial injection) were generally in the range 1.1-4.2 x 10(4) in twice-injected males and the range 1.8-9.4 x 10(3) in once-injected males. Over the next 6-11 mo, twice-injected males' titers decreased > or = 4-fold, whereas once-injected males' titers decreased slightly (1.1 - to 1.8-fold). After 6-11 mo, anti-PH-20 titers were in the range 1.0-4.8 x 10(3), and the precise residual titer did not correlate with fertility/infertility. The results show that immunization of males with PH-20, even at low doses, results in a reproducible, completely effective contraceptive action.

Subcellular distribution of protein kinase C alpha and betaI in bovine spermatozoa, and their regulation by calcium and phorbol esters.

Abstract: Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, is central to many signal transduction pathways. Previously we have demonstrated the presence of PKC in ram and bovine spermatozoa. However, the relative distribution of various PKC isozymes in the cytosolic and membrane fractions and their regulation by calcium and phorbol esters have not been elucidated. Immunocytochemical studies and Western blotting with antibodies specific for individual isoforms revealed that at least two PKC isoforms, cPKC α and cPKC β I, are found in bovine sperm cells. We demonstrate, by Western blotting analysis, that both PKC isozymes were predominantly localized in the cytosol when subcellular fractionation was carried out in the presence of EGTA. When cell lysis was carried out in the presence of Ca 2+ , most PKC α and PKC β I redistributed to the particulate fraction. Treatment of sperm cells with the biologically active phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) resulted in a rapid and extensive translocation of cytosolic PKC α and cytosolic PKC β 1 to the membrane fraction within 1 min. Furthermore, PKC's total activity was measured as a calcium- and phospholipid-dependent phosphorylation of a synthetic peptide in the cytosolic and membrane fractions derived from control and TPA-treated spermatozoa. TPA evoked a decrease in cytosolic PKC activity, accompanied by an increase in the activity associated with the plasma membrane fraction. This translocation of PKC enzymes may ensure their binding to intracellular receptor proteins (RACKs) and the phosphorylation of specific substrates, which appears to determine their physiological function. The presence of RACK in the membrane fraction of bovine sperm cells was confirmed with use of an antibody directed against the RACK protein. Previously we demonstrated the involvement of PKC in sperm acrosomal exocytosis, a process induced by signal transduction events. Thus, our results suggest that the rapid association of PKC α and PKC β I with the sperm plasma membrane, as shown in the present work for the first time, may be an early event in sperm cell regulation, leading to acrosomal exocytosis and fertilization.

Partition of the organochlorine insecticide lindane into the human sperm surface induces membrane depolarization and Ca2+ influx.

Abstract: The effects of the insecticide lindane (the gamma-isomer of 1,2,3,4,5,6-hexachlorocyclohexane) on membrane potential, cytosolic free Ca2+ concentration ([Ca2+]i) and surface biophysical properties were studied in human spermatozoa. The insecticide induces rapid, transient and reproducible membrane depolarization and opening of voltage-dependent Ca2+ channels leading to an increase in [Ca2+]i. In contrast with the effect in somatic cells, lindane did not affect gamma-aminobutyric acid receptor-linked Cl- currents. Ca2+ and K+ currents were found to drive lindane-induced membrane depolarization and repolarization respectively, whereas Na+ and Cl- fluxes appear not to have a role in the phenomenon. The insecticide was still able to produce membrane depolarization both in the combined absence of extracellular Ca2+ and Na+ and in high-K+ buffer, suggesting that lindane alters the membrane dipole potential. In agreement with this, Laurodan and Prodan fluorescence spectroscopy revealed that lindane partition into the sperm plasma membrane lowers water molecular dynamics in the uppermost region of the membrane external leaflet, probably as the result of reordering of water dipoles. We propose that the first effect of lindane partitioning into the sperm plasma membrane is a change in the membrane dipole potential, which results in the activation of membrane-located Ca2+-influx pathways.

Sperm maturation in rhesus monkey: changes in ultrastructure, chromatin condensation, and organization of lipid bilayer.

Abstract: Background The changes in ultrastructure, lipid organization, chromatin decondensation, and denaturation of rhesus monkey spermatozoa during epididymal maturation were studied This study would provide background information that would be useful to evaluate adverse effects, if any, caused by the use of contraceptive agents Methods Adult sexually mature rhesus monkeys were castrated under ketamine anesthesia The epididymis was divided into initial segment, caput, corpus, and cauda epididymides To study changes in lipid organization of the sperm plasma membrane during epididymal transit, spermatozoa from different epididymal segments and ejaculated spermatozoa were exposed to merocyanine 540 (MC 540) The changes in chromatin denaturation and decondensation were assessed by using the nucleic acid-specific fluorochromes, acridine orange, and ethidium bromide, respectively, prior to and after exposure to dithiothreitol (DTT) Results Testicular spermatozoa (∼40%) showed localization of MC 540 mainly in the midpiece, whereas remaining sperm did not localize MC 540 Spermatozoa from the initial segment of the epididymis showed uniform distribution of MC 540 localization in the head and midpiece A pattern of localization of MC 540 similar to mature caudal and ejaculated sperm in which the staining was restricted to the acrosome and the midpiece first appeared in a small percentage of caput spermatozoa and was completed during transit through the corpus epididymidis Mature spermatozoa from cauda epididymidis, vas deferens, and ejaculate did not undergo chromatin denaturation even after exposure to 10 mM DTT, unlike sperm from testis, initial segment, and caput epididymidis Spermatozoa exposed to DTT showed chromatin decondensation; maximum decondensation was seen in testicular sperm and a decrease in the percentage of sperm, showing decondensation, occurred during epididymal transit Ultrastructural studies showed that spermatozoa undergo structural changes during sperm maturation Conclusions The present study shows that rhesus monkey spermatozoa undergo reorganization of the plasma membrane lipids and stabilization of disulfide linkages during epididymal transit The results would be of use in evaluating the action of potential male contraceptive drugs on epididymal spermatozoa Anat Rec 247:25–32 © Wiley-Liss, Inc

The 26 kD protein recognized on rat cauda epididymal sperm by monoclonal antibody 4E9 has internal peptide sequence that is identical to the secreted form of epididymal protein E1

Abstract: MAb 4E9, raised against a detergent extract of rat cauda epididymal sperm, recognizes a 26 kD glycoprotein that is found on the plasma membrane of the sperm tail in cauda, but not caput, sperm (Moore et al., 1994). It also recognizes an epididymis-secreted protein that has been shown to be protein E (Xu and Hamilton, 1996). It is felt that the secreted protein becomes associated with sperm, but there has been no biochemical evidence of molecular identity between the secreted and membrane proteins. In this report, the membrane form of the antigen has been purified by reverse phase HPLC. Cyanogen bromide cleavage of the purified protein yielded 3 peptides that were purified, also by reverse phase HPLC. One of the peptides yielded an unambiguous sequence of 34 amino acids that is identical to an internal peptide of the protein found in epididymal fluid. This is the first report showing sequence identity between an epididymis-secreted protein and a protein of the sperm plasma membrane.

Fertilization and development of mouse oocytes injected with membrane-damaged spermatozoa.

Abstract: The objective of this study was to investigate the influence of sperm plasma membrane on fertilization and development in a mouse model. The sperm plasma membrane was destroyed by exposure to Triton X-100 prior to intracytoplasmic sperm injection (ICSI). A single sperm curling (SSC) test was used to evaluate cell viability. The fertilization rates of oocytes obtained following ICSI of membrane-damaged sperm was not significantly higher than that of the control group (62.4 versus 59.6%). Rates of development to blastocyst were also not significantly different (51.7 and 50%). Inner cell mass (ICM) and total embryo cell numbers in the two groups were not statistically different (16 +/- 3.7 versus 14.73 +/- 3.35 and 45.8 +/- 12.5 versus 39.5 +/- 12 respectively). There were no differences in the implantation and live fetus rates between the two groups after transfer to pseudopregnant mice (61.5 and 35.9% respectively for the membrane-damaged group and 53.5 and 31.4% for the intact group). In conclusion, the present study clearly shows that destruction of the sperm plasma membrane does not affect fertilization and further development following injection of membrane-damaged spermatozoa into mouse oocytes. Fertilization and development can be achieved by dead spermatozoa at an early stage of necrosis when only the plasma membrane has been damaged.

Isolation and characterization of a protein with homology to angiotensin converting enzyme from the periacrosomal plasma membrane of equine spermatozoa

Abstract: The periacrosomal plasma membrane of spermatozoa is involved in sperm binding to oviductal epithelial cells and to the zona pellucida. A protein of 68–70 kD molecular mass was purified biochemically from the isolated periacrosomal plasma membrane of equine spermatozoa as a possible receptor for adhesion of spermatozoa to oviductal epithelial cells. A polyclonal antibody raised in rabbits against the purified equine sperm membrane protein recognized the 70 kD and an antigenically related 32 kD protein in preparations of isolated periacrosomal sperm plasma membrane and in detergent extracted ejaculated and epididymal spermatozoa. A larger protein (∼110 kD) was detected in equine testis. Two antigenically related proteins (64 and 45 kD) were recognized on the plasma membrane of cynomolgus macaque spermatozoa. In vitro sperm-binding assays were performed in the presence of antigen-binding fragments or IgG purified from the polyclonal antiserum to investigate a possible function of the isolated protein in binding of equine spermatozoa to homologous oviductal epithelial cells or zona pellucida. Incubation with antigen-binding fragments or IgG purified from the antiserum did not inhibit binding of equine spermatozoa either to oviductal epithelial cells or to the zona pellucida. On ultrastructural examination, the antibody bound exclusively to the cytoplasmic side of the periacrosomal plasma membrane of equine and macaque spermatozoa. Microsequence analysis of 13 residues of sequence showed strong homology with a number of angiotensin converting enzymes: An 84% identity was identified with testis specific and somatic forms of human and mouse angiotensin-converting enzyme. Immunocytochemistry and immunoblot analysis established that the protein is specific for the periacrosomal membrane of ejaculated, epididymal, and testicular stallion spermatozoa. Mol. Reprod. Dev. 48:251–260, 1997. © 1997 Wiley-Liss, Inc.

Cellular Mechanisms During Mammalian Fertilization

Abstract: The sections in this article are: 1 General Introduction 2 Oogenesis and Spermatogenesis 2.1 Oogenesis: Primordial Germ Cells to Eggs 2.2 Spermatogenesis: Primordial Germ Cells to Spermatozoa 3 Final Preparation of Gametes for Fertilization 3.1 Eggs: “Meiotic Maturation” 3.2 Regulatory Aspects of Oocyte Maturation 3.3 Sperm: “Capacitation” 3.4 Sperm Activity States 4 Binding of Sperm to Eggs 4.1 Structure of the Zona Pellucida 4.2 Identification of a Mammalian Sperm Receptor 4.3 Other Mammalian Sperm Receptors 5 The Acrosome Reaction 5.1 Anatomy of the Acrosome 5.2 Stages of Exocytosis 5.3 Functions of the Acrosome Reaction during Fertilization 5.4 Site of the Acrosome Reaction 5.5 Mechanisms of the Acrosome Reaction 5.6 Initiators of Exocytosis 5.7 Conclusions 5.8 Signaling at the Sperm Plasma Membrane 5.9 Mediators of Receptor-Activated Second-Messenger Production in Sperm 5.10 Second-Messenger Production 5.11 Ca2+ and Ca2+ Channels 5.12 Internal pH 5.13 Downstream Effectors of Receptor Activation 5.14 Gamete Fusion and Cortical Granule Exocytosis 5.15 Cortical Granules 5.16 Gamete Membrane Fusion and the Receptor Question 5.17 From Oolemma to Cortical Granule—Signal Transduction Pathways

Identification of sperm plasma membrane proteins exhibiting binding affinity for the ascidian egg coat.

Abstract: In the initial stage of ascidian fertilization sequential sperm-egg coat interactions assure successful species-specific fertilization. Sperm recognize, bind to, and then penetrate the egg investment that consists of follicle cells (FC) and an acellular vitelline coat (VC). To identify plasma proteins that recognize the egg coat, a membrane fraction was prepared from Phallusia mammillata sperm using nitrogen cavitation followed by three centrifugation steps. The purity of the membrane fractions was assessed by transmission electron microscopy and marker enzymes. Comparison of the electrophoretic pattern of sperm extracellular membrane domains labeled by radio-iodination or biotinylation and recorded by autoradiography or enhanced chemiluminescence, respectively, showed the non-radioactive procedure to be a convenient and efficient method. Isolated sperm membrane components were found to inhibit fertilization in a concentration-dependent manner and to bind mainly to the FC. Eggs were used as an affinity matrix to determine which of the solubilized sperm membrane proteins possess egg-binding activity. Three biotinylated proteins (66 kDa, 120 kDa and 140 kDa) were found to bind to the VC. Assays probing heterospecific binding to Ascidia mentula eggs revealed that the 120 kDa protein possesses species-specific binding activity. Thus, the current data suggest the 120 kDa sperm membrane protein as a candidate adhesion molecule with a possible role in gamete binding and species-specific recognition in P. mammillata.

Possible Participation of a cAMP Regulated K+ Channel from the Sea Urchin Sperm in the Speract Response

Abstract: Ion channels are key elements in the activation of respiration and motility, in chemotaxis, and in triggering the acrosome reaction (AR) in sperm (Schackmann, 1989; Ward and Kopf, 1993; Darszon et al., 1994). The small size of these cells (head diameter ∼ 2 μm) has precluded the careful characterization of their electrophysiological properties. Because of this we have used planar bilayers with incorporated sperm plasma membrane components, to detect single channels from sea urchin sperm. Although with a low success rate we have also succeeded in patch clamping sea urchin sperm (Guerrero et al., 1986; Babcock et al., 1992). Using these techniques, together with studies of membrane potential, [Ca2+]i and pHi in whole sperm, we have established the presence of K+, Ca2+ and Cl- channels in this cell and are exploring their regulation and participation in chemotaxis and in the AR (Darszon et al., 1994).

The lipid and fatty acid composition of semen in relation to fertility in the male animal

Abstract: Spermatozoa are highly specialised cells which display a range of unique features associated with their crucial function of egg fertilisation. One of the most striking characteristics of spermatozoa, in biochemical terms, is the extremely high proportion of long chain highly polyunsaturated fatty acids present as components of the plasma membrane phospholipids. This high degree of unsaturation is almost unique amongst animal cells; the only other cell types which display similar levels of these polyunsaturates are the neurons of the brain and retina. The reason why spermatozoa exhibit such an unusual fatty acid composition is not clear but it is feasible that the highly unsaturated phospholipids may confer a high degree of flexibility on the sperm plasma membrane as well as provide a potential energy source in order to facilitate the characteristic flagellar motion of these cells. There is also evidence that spermatozoa lipids play a crucial role in the membrane fusion and signal transduction events associated with the acrosome reaction and fertilisation. Initial observations were made between the semen of domestic cockerel and bull with respect to animal ageing and semen quality. Within both species similar patterns were observed in that with age there was a loss of the long chain C20 and C22 polyunsaturated fatty acids accompanied by a loss of the major antioxidant enzyme systems. A decrease in phosphatidyl ethanolamine and an increase of phosphatidyl choline with age were also observed to be associated with a reduction in semen quality parameters and fertility in both species. In contrast cockerels displayed significant increases in spermatozoa and seminal plasma lipid levels where the bull exhibited losses, reflecting possible differences in spermatozoa metabolism and function.